The most serious sequela of progressive renal disease is glomerular scarring and sclerosis. Glomerular sclerosis represents an abnormal accumulation of normal glomerular basement membrane (GBM) components. Laminin is one of the normal components of GBM that accumulates within glomeruli during the progression of immunologically-mediated glomerular diseases or diabetes. Interleukin-1 (IL-1) is a ubiquitous cytokine produced in the glomeruli by both invading macrophages and by resident mesangial cells. Excessive production of IL-1 or other growth factors is thought to activate glomerular cells resulting in cell proliferation and abnormal accumulation of extracellular matrix components. although progress has been made in our understanding of the biology of glomerular lesions, little is known about intracellular molecular events that mediate production of extracellular matrix components. Our observation that in glomerular cells IL-1 increases accumulation of laminin B2 chain mRNA and stimulates the laminin B2 chain gene promoter provides an opportunity to study the intracellular molecular mechanisms involved in the normal and abnormal production of one of the components of GBM by a glomerular cell. The objective of this proposal is to identify transcription factors that control laminin B2 gene expression in glomerular cells. Through the use of comparative deletion analysis in rat and human glomerular cells, we will first attempt to identify enhancer elements required for the constitutive and IL-1-inducible activity of the laminin B2 chain gene promoter. First, rat glomerular epithelial and mesangial cells will be transfected with plasmids containing either the wild type or mutant rat laminin B2 promoter linked to a reporter gene. Transcriptional elements contained within the rat laminin B2 promoter will be identified by comparing activity of the reporter gene in rat epithelial and mesangial cells transfected in with these recombinant plasmids. Second, we will test in human mesangial cells human laminin B2 promoter constructs with deletion of homologous regions contained and transcriptionally active in the rat promoter. Comparison of deletion mutants in the rat and human systems should facilitate localization of sequences transcriptionally active and homologous in the promoters from the two species. Based on the information from the comparative deletion analysis of the human and rat laminin B2 promoter, we will use specific DNA probes in electrophoretic gel shift assays (EMSA), Southwestern blots and methylation interference to identify constitutive and IL-1-inducible transcription factors that regulate the activity of the laminin B2 promoter. Protein(s) that we identify as novel and/or specific for glomerular cells will be purified using sequential anion exchange and tandem DNA-affinity chromatography. Partial amino acid sequences of this protein(s) will be used to synthesize degenerate oligonucleotide primers for polymerase chain reaction (PCR). cDNA libraries derived from glomerular cells will be screened with the PCR-amplified probes to clone the laminin B2 promoter-specific transcription factor(s). Identification of transcription factors involved in the rat and human laminin B2 gene expression would set the ground work for future studies on transcriptional processes of laminin production in intact glomeruli during development and in the course of glomerular disease.